This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. In the corpus luteum, cholesterol transport to the mitochondrial P450 side chain cleavage enzyme (P450scc) is thought to be the rate limiting step in steroid production. The mechanism for moving cholesterol to the P450scc is uncertain. Since cholesterol is insoluble in the cell, it must be transported to the P450scc while bound to a carrier molecule. Sterol carrier protein-2 (SCP2) is a 13.2 kDa protein believed to facilitate the transport of cholesterol to the mitochondria. SCP2 is reported to be synthesized as a 15.3 kDa pro-form with an N-terminal region which is thought to target SCP2 to the mitochondria. The C- terminal SCP2 sequence may, however, also target this protein to the peroxisome. Since the encoded SCP2 amino acid sequence may direct this protein to separate locations, other post-translational mechanisms must exist which modify SCP2 and govern its final destination. Altered cholesterol transport to the mitochondrial has recently been identified as a key lytic event in the corpus luteum, and the long term goal of this proposal is to focus on the potential negative regulatory actions of PGF2alpha on SCP2 function during luteolysis. We hypothesize that PGF2alpha reduces cAMP-mediated SCP2 gene expression, and activates PKC- mediated post-translational SCP2 phosphorylation. These activities may alter SCP2 expression and function, thereby reducing intracellular cholesterol transport capacity which leads to a decline in steroid production. To test this hypothesis, the experiments outlined in this proposal will 1) determine whether PGF2alpha alters basal or gonadotropin (LH/hCG) induced SCP2 expression and localization in the corpus luteum, 2) determine whether PGF2alpha alters luteal steroidogenesis through PKC mediated SCP2 phosphorylation, 3) determine whether pro-SCP2 or mature SCP2 is targeted to the mitochondria and determine whether phosphorylation of either SCP2 form alters its capacity to mediate steroidogenesis, 4) determine whether PGF2alpha alters the expression of specific PKC isoforms in the corpus luteum and determine whether this isoform mediates SCP2 phosphorylation and 5) determine whether estradiol alters PGF2alpha/PKC mediated SCP2 phosphorylation and expression in the corpus luteum. The investigation outlined in this proposal will provide a unique opportunity to study an essential hormone/prostaglandin mediated process in endocrine cell physiology. This research will also provide important new information concerning SCP2 regulation, expression and function during luteal regression.